Chlamydia trachomatis is the etiological agent of several significant diseases of humans including trachoma, the leading cause of infectious blindness worldwide. It is also the most common cause of sexually transmitted disease in the USA. Other species of medical importance include C. pneumoniae, a causative agent of upper respiratory tract infections and possibly associated with atherosclerosis, and C. psittaci, which is primarily a pathogen of animals but occasionally is transmitted to humans. Chlamydiae are obligate intracellular bacteria that undergo their life cycles entirely within an intracellular vesicle that is isolated from established routes of intracellular vesicle trafficking. Whereas the majority of intracellular parasites are thought to block maturation of the endocytic vesicle to a lysosome, chlamydiae dissociate themselves from this pathway and establish a functional interaction with an exocytic pathway which delivers sphingolipids from the Golgi apparatus to the plasma membrane. Interaction with this secretory pathway provides a novel pathogenic mechanism allowing chlamydiae to establish themselves in a site not destined to fuse with lysosomes. Chlamydial avoidance of lysosomal fusion appears to occur in two stages. Upon endocytosis by eukaryotic cells, the nascent inclusion is non-fusogenic with any endocytic or exocytic pathway. A second, active phase of lysosomal avoidance requires chlamydial protein synthesis to modify the properties of the inclusion. One of the initial events in chlamydial infection is the expression of a chlamydial gene product(s) that effectively isolates the inclusion from the endosomal/lysosomal pathway and initiates fusion competence with a subset of exocytic vesicles but with minimal interference of normal cellular function. Chlamydiae, like many Gram-negative pathogenic bacteria, subvert the responses of the eukaryotic host by the secretion of specific effector proteins directly into the cytosol of the host cell using a specific transport system known as type III secretion to inject bacterial proteins into the host cell. Chlamydiae possess the components of a complete type III secretion system although the identify and function of the secreted effector proteins and even when they are secreted are unknown. Over the past year, we have shown that chlamydiae secrete at least two distinctt classes of effector proteins throughout the developmental cycle. One protein is pre-exisitng in elementary bodies and is secreted into the host cell where it is recognized by the host and serves to trigger the recruitment of actin to promote internalization. Other secreted effector proteins are not synthesized until the chlamydiae are intracellular. Several of these latter proteins are known to be inserted into and modify the vacuolar (inclusion) membrane which encompasses the replicating chlamydiae. These results show potential not only for defining the interactions of chlamydiae with the host cell but will serve as a model system for other obligate intracellular pathogens which occupy vacuoles that do not fuse with lysosomes. Understanding the initial events in chlamydial differentiation including the transition in properties of the endocytic vesicle to one which intersects an exocytic pathway remain significant challenges in defining the pathogenic mechanisms of chlamydiae. Additional studies have focused upon the early signals to intiate chlamydial development from the metabolically dormant elementary bodies to the a replicative form. A key step in this process is the dissociation of the chlamydial chromatin from a condensed state maintained by the presence of histone-like proteins. Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1. Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active although the mechanisms of Hc1 release from DNA have remained unknown. Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1. A genetic screen was therefore designed to identify chlamydial genes rescuing E. coli from the lethal effects of Hc1 overexpression. CT804, a gene homologous to ispE, an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was selected. E. coli co-expressing CT804 and Hc1 grew normally although they expressed Hc1 to a level equivalent to that which condensed the chromatin of parent Hc1-expressing controls. Inhibition of the MEP pathway with fosmidomycin abolished IspE rescue of Hc1-expressing E. coli. Deproteinated extract from IspE expressing bacteria caused dispersal of purified chlamydial nucleoids suggesting that chlamydial histone-DNA interactions are disrupted by a small metabolite within the MEP pathway rather than by direct action of IspE. By partial reconstruction of the MEP pathway, we determined that 2C-methyl-erythritol-2,4-cyclodiphosphate dissociated Hc1 from chlamydial chromatin. These results suggest that chlamydial histone-DNA interactions are disrupted upon germination by a small metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis. Interactions of histones and DNA-binding proteins with DNA to establish chromatin structure are regulated by many mechanisms including covalent modification by phosphorylation, methylation, or acetylation, proteolysis, or topology of the DNA itself. Disruption of chlamydial Hc1-DNA interactions via a small metabolite is an unusual means of releasing histone from chromatin. Release of chlamydial chromatin from the constraints imposed by Hc1 is an essential early event in the chlamydial developmental cycle. That chlamydiae utilize an intermediate in a metabolic pathway not present in mammalian cells suggests a potential target for development of chemotherapeutic agents that could inhibit this critical event.